Project 2: James Cunningham, M.D. Ebola virus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality, and lack of effective vaccine or anti-viral therapy have created a high level of public concern about EboV. EboV are enveloped viruses that enter cells by attachment of envelope glycoprotein GP to cell surface lectins and uptake by macropinocytosis. Virus-containing vesicles fuse with endosomes, and virus particles are transported to lysosomes containing host factors that activate the intrinsic membrane fusion activity of the EboV glycoprotein GP. The goal of this proposal is to identify small molecules that inhibit infection mediated by EboV GP and to use these inhibitors as tools to investigate the virus entry pathway. Previously, a small molecule library was screened using a cell-based assay for infection mediated by the EboV glycoprotein GP. Three compounds have been identified that exhibit nanomolar anti-EboV activity, dynamic structure-activity profiles, absence of cytotoxicity, and evidence of a unique target in cells. The Specific Aims of this project are to use iterative SAR to enhance the potency and the pharmacokinetic and pharmacokinetic properties of the inhibitors and to develop derivatives to use as probes of the Ebov entry pathway. New methods will be developed to identify the targets of these inhibitors in cells and to determine the role ofthe target in infection. This information will be utilized to determine the mechanism and consequences of inhibitor action and to test the efficacy of the most promising inhibitors in a mouse model of infection. Preliminary studies reveal the target of one ofthe inhibitors as the lysosomal membrane protein Niemann-Pick Cl (NPC1) and demonstrate that the inhibitor interferes with the function of NPC1 as an EboV GP receptor. These findings validate the use of a chemical biology approach for analysis of the EboV entry pathway. To carry out these studies, a team of investigators has been assembled with complementary expertise in organic synthesis, medicinal chemistry, quantitative mass spectrometry, genetic screening for virus host factors, and mouse models of EboV infection in BL4 laboratory at USAMRIID, Ft Detrick, MD.